Oxygen carriers that are useful as oxygen therapeutics (sometimes referred to as “oxygen-carrying plasma expanders”) can be grouped into three categories: i) perfluorocarbon-based emulsions; ii) liposome-encapsulated hemoglobin (“Hb”); and iii) modified Hb. As discussed below, none of these therapeutics have been entirely successful. However, products comprising modified cell-free Hb are thought to be the most promising. Perfluorochemical-based emulsions must be emulsified with a lipid, typically egg-yolk phospholipid, before they can be used in biological systems. Unlike Hb, these emulsions dissolve molecular oxygen rather than binding the oxygen as a ligand. Though the perfluorocarbon emulsions are inexpensive to manufacture, they do not carry sufficient oxygen at clinically tolerated doses to be effective. Conversely, while liposome-encapsulated Hb has been shown to be effective, it is too costly for widespread use. (See generally, Winslow, R. M., “Hemoglobin-based Red Cell Substitutes,” Johns Hopkins University Press, Baltimore (1992)).
Initial attempts to use free Hb from erythrocyte hemolysates as a red cell substitute were unsuccessful. The stromal components were found to be toxic, resulting in coagulopathy associated with acute renal failure. In 1967, the first stroma-free Hb (“SFH”) solutions had been prepared for use as plasma expanders (Rabiner, S. F. et al., 1967, J. Exp. Med. 126:1127-1142). However, these solutions had a limited transfusion half-life of about 100 minutes.
The reason for the short circulation half-life of SFH is due to the ability of the tetrameric protein to dissociate into dimers, which are rapidly filtered from the circulation by the kidneys. Accordingly, a multitude of methods for cross-linking Hb to retain its tetrameric structure were devised. U.S. Pat. No. 5,296,465 describes intramolecular cross-linking to prevent dimer formation. Cross-linking Hb may be achieved with diaspirin (diesters of bis-3,5-dibromosaliocylate, U.S. Pat. No. 4,529,719) or 2-N-2-formyl-pyridoxal-5′-phosphate and borohydride (Benesch, R. E. et al., 1975, Biochem. Biophys. Res. Commun. 62:1123-1129). In addition, Simon, S. R. and Konigsberg, W. H. (1966, PNAS 56:749-56) disclosed the use of bis-(N-maleimidomethyl)ether (“BME”) to generate intramolecularly cross-linked Hb that was reported to have a four-fold increase in half-life when infused into rats and dogs (Bunn, H. F. et al., 1969, J. Exp. Med. 129:909-24). However, the cross-linking of Hb with BME resulted in the concomitant increase in the oxygen affinity of Hb, which at the time was thought to prevent its use as a potential HBOC.
In addition, methods for conjugating Hb to macromolecules were developed to increase hydrodynamic size and limit or prevent extravasation. Cross-linking SFH to form poly-Hb is described in U.S. Pat. Nos. 4,001,200 and 4,001,401. SFH was also linked to other macromolecules such as dextran (Chang, J. E. et al., 1977, Can. J. Biochem. 55:398-403), hydroxyethyl starch (DE 2,161,086), gelatin (DE 2,449,885), albumin (DE 2,449,885) and PEG (DE 3,026,398 and U.S. Pat. Nos. 4,670,417, 4,412,989 and 4,301,144).
Some of the physiological effects of these oxygen-carrying solutions are not fully understood. Perhaps the most controversial effect is the propensity to cause vasoconstriction, which can manifest as hypertension in animals and man (Amberson, W., 1947, Science 106:117-117 and Keipert, P. et al., 1993, Transfusion 33:701-708). One of the first red cell substitutes developed by the U.S. Army was human Hb cross-linked between α-chains with bis-dibromosalicyl-fumarate (“ααHb”). However, ααHb was abandoned after it showed severe increases in pulmonary and systemic vascular resistance (Hess, J. et al., 1991, Blood 78:356A). A commercial version of this cross-linked Hb was also abandoned after a disappointing Phase III clinical trial (Winslow, R. M., 2000, Vox Sang 79:1-20).
The most commonly advanced explanation for the vasoconstriction produced by cell-free Hb is that it readily binds the endothelium-derived relaxing factor, nitric oxide (“NO”). Two molecular approaches have been advanced in attempting to overcome the NO binding activity of Hb. The first approach attempted to reduce the NO binding by modifying the distal heme pocket through site-specific mutagenesis (Eich, R. F. et al., 1996, Biochem. 35:6976-83). The second approach attempted to reduce or inhibit extravasation of Hb by increasing its molecular size (Hess, J. R. et al., 1978, J. Appl. Physiol. 74:1769-78, Muldoon, S. M. et al., 1996, J. Lab. Clin. Med. 128:579-83, Macdonal, V. W. et al., 1994, Biotechnology 22:565-75, Furchgott, R., 1984, Ann. Rev. Pharmacol. 24:175-97 and Kilbourne, R. et al., 1994, Biochem. Biophys. Res. Commun. 199:155-62).
Recombinant Hbs with reduced NO affinity have been produced that are less hypertensive in top-load rat experiments (Doherty, D. H. et al., 1998, Nature Biotechnology 16:672-676 and Lemon, D. D. et al., 1996, Biotech 24:378). However, studies suggest that NO binding is not the only explanation for the vasoactivity of Hb. Specifically, certain large Hb molecules, such as those modified with PEG, were virtually free of the hypertensive effect, even though their NO binding rates were identical to those of ααHb (Rohlfs, R. J. et al., 1998, J. Biol. Chem. 273:12128-12134). In addition, PEG modified Hb was extraordinarily effective in preventing the consequences of hemorrhage when given as an exchange transfusion prior to hemorrhage (Winslow, R. M. et al., 1998, J. Appl. Physiol. 85:993-1003).
The conjugation of PEG to Hb reduces antigenicity and extends circulation half-life of the Hb. However, the PEG-conjugation reaction has been reported to result in dissociation of Hb tetramers into monomer subunits. These low molecular weight monomeric PEG-Hb conjugates caused gross hemoglobinuria when transfused into rats (Iwashita and Ajisaka, Organ-Directed Toxicity Chem. Indices Mech., Proc. Symp., Brown et al., 1981, Eds. Pergamon, Oxford, England pgs 97-101). A polyalkylene oxide (“PAO”) conjugated Hb, having a molecular weight greater than 84,000 Da, was prepared by Enzon, Inc. (U.S. Pat. No. 5,650,388). The conjugate contained ten PEG-5,000 chains linked to Hb at its α- and ε-amino groups. This degree of substitution was described as avoiding hemoglobinuria and associated nephrotoxicity in mammals. However, the conjugation reaction resulted in a heterogeneous conjugate population and contained other undesirable reactants that had to be removed by column chromatography.
PEG conjugation is typically performed by reacting activated PEG with functional groups on the surface of biomolecules. The most common functional groups in a biomolecule are: the amino groups of lysine and histidine residues, thiol groups of cysteine residues, and the hydroxyl groups of serine, threonine and tyrosine residues. The N- and C-termini of the biomolecule may also act as active functional groups. PEG is usually activated by converting the hydroxyl terminus to a moiety capable of reacting with these functional groups in a mild aqueous environment. One of the most common PEGs used for conjugation of therapeutic biopharmaceuticals is methoxy-PEG (“mPEG”). Methoxy-PEG has only one functional group (i.e. hydroxyl), which minimizes cross-linking and aggregation problems that are associated with bifunctional PEG. However, mPEG is often contaminated with high molecular weight PEG (i.e. “PEG diol”). The contamination problem is the result of the production process and is further aggravated as the molecular weight of the PEG increases. In mPEG the amount of contaminant can range as high as 10 to 15% (Dust, J. M. et al., 1990, Macromolecule 23:3742-3746) due to its production process. The purity of mPEG is especially critical for the production of PEG conjugated biotherapeutics because the FDA requires a high level of reproducibility in the production processes and quality of the final product.
A variety of linkers and methods have been developed for conjugating macromolecules to Hb. Phenyl linkers, such as 4-phenylmaleimido or 3-phenylmaleimido (U.S. Pat. No. 5,750,725) and isothiocyanophenylcarbamate (U.S. Pat. No. 7,144,989) have been used to conjugate PEG-5,000 to Hb. However, the use of phenyl groups in a blood substitute is believed by some to be undesirable. To avoid the use of phenyl linkers, succinimidyl activated PEG was prepared for binding free ε-amines available on the surface of Hb (Larwood, D. J. and Szoka, F. C., 1984, J. Labeled Compounds Radiopharm. 21:603-14). However, the ester bond formed between PEG and the succinimidyl group is easily hydrolyzed in the body. To address this issue, activated PAOs having reactive moieties that produce urethane linkages with ε-amino groups of Hb were developed. The urethane linkages are less susceptible to hydrolytic degradation in the circulatory system (U.S. Pat. No. 5,234,903). Other methods have been utilized that employ thiolation of the ε-amines of Hb for binding maleimide activated PAOs (U.S. Pat. No. 6,844,317). The thioester bonds formed under these methods are less susceptible to degradation (U.S. Pat. App. No. 2006/0135753).
Conjugation of Hb to PAOs has been performed in both the oxygenated and deoxygenated states. U.S. Pat. No. 6,844,317 describes conjugating Hb in the oxygenated, or “R” state, to enhance the oxygen affinity of the resultant PEG-Hb conjugate. This is accomplished by equilibrating Hb with the atmosphere before conjugation. Others describe a deoxygenation step prior to conjugation to diminish the oxygen affinity and increase structural stability. The increased stability enables the Hb to withstand the physical stresses of chemical modification, diafiltration and/or sterile filtration and sterilization (U.S. Pat. No. 5,234,903). Deoxygenating Hb prior to modification has also been suggested for efficient intramolecular cross-linking. U.S. Pat. No. 5,234,903 discloses that deoxygenation is required to expose lysine 99 of the α-chain for intramolecular cross-linking.
Stability of HBOCs for storage is currently under investigation. Susceptibility to autoxidation and the formation of MetHb may hinder the clinical use of HBOCs. Formation of MetHb in the circulation can be deleterious for a number of reasons. MetHb does not bind oxygen. Consequently, its formation decreases the oxygen carrying capacity of blood. The presence of MetHb in the blood gives rise to reactive oxygen species “ROS”, which may play a role in the development of oxidative damage in vivo (Reeder, B. J. et al., 2004, Antioxid. Redox Signal 6:941-943). In addition, the chemical bond that stabilizes the heme in native Hb is weakened in MetHb. This destabilization allows the heme to be released more quickly, thereby giving rise to a cascade of effects, including induction of heme oxygenase-1 (Balla, G. et al., 1993, PNAS 90:9280-9284 and Motterlini, R. et al., 1995, Am. J. Physiol. 269:H648). In whole blood, oxidation is made reversible by an effective redox system catalyzed by NADPH-dependent MetHb reductase. Unfortunately, isolated Hb does not benefit from this defense mechanism. It has been shown, however, that red blood cells in vitro and in the presence of ascorbate have reduced the formation of extracellular MetHb (McGown, E. L. et al., 1990, Biochem. Biophys. Acta 1036:202-206).
In contrast, it was recently found that the antioxidant ascorbate rapidly oxidized PEG-Hb (Vandegriff, K. D. et al., 2006, Biochem. J. 399:463-471). Other investigations have used antioxidants to stabilize polymerized Hb compositions under deoxygenated conditions (U.S. Pat. Nos. 5,895,810 and 7,435,795). However, to date, antioxidants have not been successful in reducing or preventing auto-oxidation of HBOC's.
Accordingly, there is a need for a method of preparing deoxygenated PEG-Hb conjugates having reduced autoxidation rates when stored at low or ambient temperatures.